Circ-PGPEP1 augments renal cell carcinoma proliferation, Warburg effect, and distant metastasis.

Circular RNAs (circRNAs) contribute to the malignant phenotype and progression of several types of human cancers, including renal cell carcinoma (RCC). This study probed the molecular mechanism of circPGPEP1 regulating RCC proliferation, Warburg effect, and distant metastasis by targeting the miR-378a-3p/JPT1 axis. Here identified higher circPGPEP1 expression in RCC tissues and cells by RT-qPCR, and high levels of circPGPEP1 were positively correlated with high histological grade and distant metastasis in RCC patients. Furthermore, patients with high levels of circPGPEP1 had a worse survival prognosis. Functional assays presented that knockdown of circPGPEP1 inhibited RCC proliferation, invasion, migration, EMT, and Warburg effect. Dual-luciferase reporter assay, RNA immunoprecipitation, nucleoplasmic RNA isolation, and functional rescue experiments confirmed that circPGPEP1 induced JPT1 expression by sponging miR-378a-3p, thereby promoting RCC malignant phenotype. Xenograft assays and metastasis models further demonstrated that down-regulation of circPGPEP1 effectively inhibited tumor growth and distant metastasis of RCC. Taken together, circPGPEP1, a prognostic circRNA in RCC, acts through the miR-378a-3p/JPT1 axis to regulate RCC progression.

Crystal structure analysis of pyrrolidone carboxyl peptidase from Thermus thermophilus.

Pyrrolidone carboxyl peptidase (PCP) hydrolytically removes the L-pyroglutamic acid from the amino terminal region of pyroglutamyl proteins or peptides. So far, only a limited number of structures of PCP have been solved. Here we report the crystal structure of pyrrolidone carboxyl peptidase from Thermus thermophilus (TtPCP) which has been solved using the molecular replacement method and refined at 1.9 Å resolution. TtPCP follows the α/ß/α architecture in which the central ß-sheets are surrounded by α-helices on both sides. The inter subunit contact between two monomers consists of two short antiparallel ß-strands and part of a long protrusion loop. By comparing the TtPCP with its structural homologs, we identified the putative catalytic triad residues as Glu76, Cys139 and His160. A unique disulfide link found in some homologs of TtPCP, formed between two monomers that provide thermal stability to the protein, is not observed in TtPCP. Hence, being a thermophilic protein, the putative thermal stability of TtPCP could be due to more intra and inter-molecular hydrogen bonds, hydrophobic and ion pair interactions when compared with its mesophilic counterpart. The structural details of TtPCP will be helpful to understand the basis of the intrinsic stability of thermophilic proteins. Also, it could be useful for protein engineering.

Two-photon fluorogenic probe for visualizing PGP-1 activity in inflammatory tissues and serum from patients.

A PGP-1-specific one/two-photon fluorogenic probe (BH1), capable of high sensitivity, super selectivity, and visual imaging of endogenous PGP-1 activity from live mammalian cells and serum/skin tissues from patients by using one/two-photon fluorescence microscopy (O/TPFM).

A ratiometric fluorescent sensor for rapid detection of the pyroglutamate aminopeptidase-1 in mouse tumors.

Pyroglutamate aminopeptidase-1 (PGP-1) is an important enzyme that plays an indispensable role in the process of inflammation. Up to now, few reports have been reported on the detection of PGP-1 activity in vivo and in vitro, and there are no reports on ratiometric detection. Here, the first red-emitting ratiometric fluorescent sensor (DP-1) for the specific detection of PGP-1 both in vivo and in vitro was designed and synthesized by using DCD-NH2 as the luminescent parent and pyroglutamate as a recognition group. After interacting with PGP-1, the amide bond is hydrolyzed by the enzyme and the color of the solution changes from yellow (λabs = 420 nm) to red (λabs = 520 nm), accompanied by obvious fluorescence emission wavelength change (from ∼564 nm to ∼616 nm). The probe has high specificity and sensitivity towards PGP-1 in about 10 min, and the DL is as low as 0.25 ng mL-1. Interestingly, under the stimulation of Freund's incomplete adjuvant and lipopolysaccharide, the imaging of DP-1 in HepG2 and RAW264 cells shows that the expression of PGP-1 is associated with inflammation. What's more, for the first, the imaging of a mouse tumor model confirms that the enzyme is closely related to the occurrence of some inflammation and tumor diseases. These results indicate that DP-1 can be used as an effective tool for real-time monitoring of PGP-1 levels both in vivo and in vitro and the study of inflammatory tumor pathology.

A bioluminescent probe for in vivo imaging of pyroglutamate aminopeptidase in a mouse model of inflammation.

Pyroglutamate aminopeptidase (PGP) specifically cleaves the peptide bond of pyroglutamic acid linked to the N-terminal end of a polypeptide or protein. Previous studies showed that PGP was associated with several physiological processes and diseases especially those involving inflammation. Utilizing a 'caging' strategy, we designed and synthesized a bioluminescence probe (PBL) with a limit-of-detection of 3.7 * 10-4 mU/mL. In vivo imaging in a mouse model of inflammatory liver disease revealed that the probe has excellent sensitivity and selectivity and provides a powerful tool for studying the physiological and pathological processes involving PGP.

The Role of High Fat Diets and Liver Peptidase Activity in the Development of Obesity and Insulin Resistance in Wistar Rats.

High-fat diets (HFD) have been widely associated with an increased risk of metabolic disorders and overweight. However, a high intake of sources that are rich in monounsaturated fatty acids has been suggested as a dietary agent that is able to positively influence energy metabolism and vascular function. The main objective of this study was to analyze the role of dietary fats on hepatic peptidases activities and metabolic disorders. Three diets: standard (S), HFD supplemented with virgin olive oil (VOO), and HFD supplemented with butter plus cholesterol (Bch), were administered over six months to male Wistar rats. Plasma and liver samples were collected for clinical biochemistry and aminopeptidase activities (AP) analysis. The expression of inducible nitric oxide synthase (iNOS) was also determined by Western blot in liver samples. The diet supplement with VOO did not induce obesity, in contrast to the Bch group. Though the VOO diet increased the time that was needed to return to the basal levels of plasma glucose, the fasting insulin/glucose ratio and HOMA2-%B index (a homeostasis model index of insulin secretion and valuation of ß-cell usefulness (% ß-cell secretion)) were improved. An increase of hepatic membrane-bound dipeptidyl-peptidase 4 (DPP4) activity was found only in VOO rats, even if no differences in fasting plasma glucagon-like peptide 1 (GLP-1) were obtained. Both HFDs induced changes in hepatic pyroglutamyl-AP in the soluble fraction, but only the Bch diet increased the soluble tyrosyl-AP. Angiotensinase activities that are implicated in the metabolism of angiotensin II (AngII) to AngIV increased in the VOO diet, which was in agreement with the higher activity of insulin-regulated-AP (IRAP) in this group. Otherwise, the diet that was enriched with butter increased soluble gamma-glutamyl transferase (GGT) and Leucyl-AP, iNOS expression in the liver, and plasma NO. In summary, VOO increased the hepatic activity of AP that were related to glucose metabolism (DPP4, angiotensinases, and IRAP). However, the Bch diet increased activities that are implicated in the control of food intake (Tyrosine-AP), the index of hepatic damage (Leucine-AP and GGT), and the expression of hepatic iNOS and plasma NO. Taken together, these results support that the source of fat in the diet affects several peptidases activities in the liver, which could be related to alterations in feeding behavior and glucose metabolism.

Altered peptidase activities in thyroid neoplasia and hyperplasia.

BACKGROUND: Papillary thyroid carcinoma (PTC), follicular thyroid adenoma (FTA), and thyroid nodular hyperplasia (TNH) are the most frequent diseases of the thyroid gland. Previous studies described the involvement of dipeptidyl-peptidase IV (DPPIV/CD26) in the development of thyroid neoplasia and proposed it as an additional tool in the diagnosis/prognosis of these diseases. However, very little is known about the involvement of other peptidases in neoplastic and hyperplastic processes of this gland. METHODS: The catalytic activity of 10 peptidases in a series of 30 PTC, 10 FTA, and 14 TNH was measured fluorimetrically in tumour and nontumour adjacent tissues. RESULTS: The activity of DPPIV/CD26 was markedly higher in PTC than in FTA, TNH, and nontumour tissues. Aspartyl aminopeptidase (AspAP), alanyl aminopeptidase (AlaAP), prolyl endopeptidase, pyroglutamyl peptidase I, and aminopeptidase B activities were significantly increased in thyroid neoplasms when compared to nontumour tissues. AspAP and AlaAP activities were also significantly higher in PTC than in FTA and TNH. CONCLUSIONS: These data suggest the involvement of DPPIV/CD26 and some cytosolic peptidases in the neoplastic development of PTC and FTA. Further studies will help to define the possible clinical usefulness of AlaAP and AspAP in the diagnosis/prognosis of thyroid neoplasms.

Activity of soluble aminopeptidase A and dipeptidyl peptidase IV and membrane-bound aminopeptidase B and pyroglutamyl peptidase I in adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis.

OBJECTIVE: To analyze soluble and membrane-bound peptidase activities in the tonsils and adenoids removed from patients with adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis. METHODS: A total of 48 tissue samples from patients undergoing adenoidectomy and tonsillectomy for adenoid hyperplasia, tonsillar hyperplasia or chronic tonsillitis were analyzed. The catalytic activity of a pool of peptidases in the soluble (dipeptidyl peptidase IV, aminopeptidase A, aminopeptidase N and cystinyl aminopeptidase) and membrane-bound (prolyl endopeptidase, aspartyl aminopeptidase, aminopeptidase B and pyroglutamyl peptidase I) fractions was measured fluorometrically. RESULTS: The activity of membrane-bound aminopeptidase B was higher in cases of chronic tonsillitis and adenoid hyperplasia than in tonsillar hyperplasia, p=0.004. Soluble dipeptidyl peptidase IV and membrane-bound pyroglutamyl peptidase I were found to be more active in tissues from male chronic tonsillitis tissues, p<0.05, while membrane-bound aminopeptidase B activity was higher in tissues of females with tonsillar hyperplasia, p<0.001. In the case of chronic tonsillitis, soluble aminopeptidase A was found to have a higher level of activity in tissues from children than those from adults, p=0.005. CONCLUSIONS: Our results suggest a potential role of soluble aminopeptidase A, soluble dipeptidyl peptidase IV, membrane-bound aminopeptidase B and membrane-bound pyroglutamyl peptidase I in the pathobiology of adenoid hyperplasia, tonsillar hyperplasia and chronic tonsillitis that is differently regulated as a function of gender. These finfings may modify in the future the clinical approach to these diseases.

Removal of N-terminal blocking groups from proteins.

Two enzymatic methods commonly used in N-terminal sequence analysis of blocked proteins are presented: one uses pyroglutamate aminopeptidase for N(α)-pyrrolidone carboxyl-proteins in solution or blotted onto a membrane, and the other uses acylaminoacyl-peptide hydrolase for N(α)-acyl-proteins blocked with other acyl groups. A Support Protocol describes a colorimetric assay for pyroglutamate aminopeptidase activity. Sequencing with acylaminoacyl-peptide hydrolase must include fragmentation of the protein before unblocking, so procedures are provided for chemically blocking newly generated peptides with either succinic anhydride or phenylisothiocyanate/performic acid. The hydrolase is then applied to the total mixture of peptides, only one of which, the acylated N-terminal peptide, should be a substrate for hydrolase. After incubation, the mixture of peptides is subjected to sequence analysis.

Streptococcus merionis sp. nov., isolated from Mongolian jirds (Meriones unguiculatus).

Gram-positive, catalase-negative, chain-forming, coccus-shaped organisms were isolated both from intraperitoneally grown vesicles of the fox tapeworm Echinococcus multilocularis and the oropharynges of laboratory-kept Mongolian jirds (Meriones unguiculatus). The strains displayed no haemolytic activity on Columbia sheep blood agar, pyrrolidonyl arylamidase activity was negative and the organisms reacted weakly with Lancefield group D antiserum. On the basis of phenotypic characteristics, the strains were tentatively identified as members of the genus Streptococcus. Comparative 16S rRNA gene sequencing studies confirmed their assignment to the genus Streptococcus and revealed that Streptococcus hyointestinalis DSM 20770(T) was their closest phylogenetic neighbour (96.5 % sequence similarity). The levels of 16S rRNA gene sequence similarity between the isolates and representatives of species of the genus Streptococcus were only 95.7-96.2 %. On the basis of the phenotypic and molecular data presented, the isolates from Mongolian jirds represent a novel species of the genus Streptococcus, for which the name Streptococcus merionis sp. nov. is proposed. The type strain is WUE3771(T) (=DSM 19192(T)=CCUG 54871(T)).

Chronic ethanol intake modifies pyrrolidon carboxypeptidase activity in mouse frontal cortex synaptosomes under resting and K+ -stimulated conditions: role of calcium.

Pyrrolidon carboxypeptidase (Pcp) is an omega peptidase that removes pyroglutamyl N-terminal residues of peptides such as thyrotrophin-releasing hormone (TRH), which is one of the neuropeptides that has been localized into many areas of the brain and acts as an endogenous neuromodulator of several parameters related to ethanol (EtOH) consumption. In this study, we analysed the effects of chronic EtOH intake on Pcp activity on mouse frontal cortex synaptosomes and their corresponding supernatant under basal and K+ -stimulated conditions, in presence and absence of calcium (Ca2+) to know the regulation of Pcp on TRH. In basal conditions, chronic EtOH intake significantly decreased synaptosomes Pcp activity but only in absence of Ca2+. However, supernatant Pcp activity is also decreased in presence and absence of calcium. Under K+-stimulated conditions, chronic EtOH intake decreased synaptosomes Pcp activity but only in absence of Ca2+, whereas supernatant Pcp activity was significantly decreased only in presence of Ca2+. The general inhibitory effect of chronic EtOH intake on Pcp activity suggests an inhibition of TRH metabolism and an enhancement of TRH neurotransmitter/neuromodulator functions, which could be related to putative processes of tolerance to EtOH in which TRH has been involved. Our data may also indicate that active peptides and their degrading peptidases are released together to the synaptic cleft to regulate the neurotransmitter/neuromodulator functions of these peptides, through a Ca2+ -dependent mechanism.

Microbiological characteristics, presumptive identification, and antibiotic susceptibilities of Staphylococcus lugdunensis.

This study validated abbreviated methods for the presumptive identification of Staphylococcus lugdunensis and studied the antibiotic susceptibilities of 106 isolates. The combination of positive responses to ornithine and pyrrolidonyl arylamidase identified all S. lugdunensis isolates. Resistance to penicillin and methicillin was detected in 27 and 5% of isolates, respectively.

Improved recovery of proteome-informative, protein N-terminal peptides by combined fractional diagonal chromatography (COFRADIC).

We previously described a proteome-wide, peptide-centric procedure for sorting protein N-terminal peptides and used these peptides as readouts for protease degradome and xenoproteome studies. This procedure is part of a repertoire of gel-free techniques known as COmbined FRActional DIagonal Chromatography (COFRADIC) and highly enriches for alpha-amino-blocked peptides, including alpha-amino-acetylated protein N-terminal peptides. Here, we introduce two additional steps that significantly increase the fraction of such proteome-informative, N-terminal peptides: strong cation exchange (SCX) segregation of alpha-amino-blocked and alpha-amino-free peptides and an enzymatic step liberating pyroglutamyl peptides for 2,4,6-trinitrobenzenesulphonic acid (TNBS) modification and thus COFRADIC sorting. The SCX step reduces the complexity of the analyte mixture by enriching N-terminal peptides and depleting alpha-amino-free internal peptides as well as proline-starting peptides prior to COFRADIC. The action of pyroglutamyl aminopeptidases prior to the first COFRADIC peptide separation results in greatly diminishing numbers of contaminating pyroglutamyl peptides in peptide maps. We further show that now close to 95% of all COFRADIC-sorted peptides are alpha-amino-acetylated and, using the same amount of starting material, our novel procedure leads to an increased number of protein identifications.

The expression of TRH, its receptors and degrading enzyme is differentially modulated in the rat limbic system during training in the Morris water maze.

TRH administration induces arousal, improves cognition, and modulates glutamatergic and cholinergic transmission in hippocampal neurons. To study the possible involvement of TRH neurons in learning and memory processes, gene expression of TRH, its receptors, and pyroglutamyl peptidase (PPII), were measured in limbic regions of water-maze trained rats. Hypothalamus and amygdala showed changes related to the task but not specific to spatial learning while in hippocampus, pro-TRH and TRH-R1 mRNA levels were specifically increased in those animals trained to find a hidden platform. Variation of TRH content and mRNA levels of pro-TRH, TRH-R1, TRH-R2 and PPII are observed in conditions known to activate TRH hypophysiotropic neurons. Changes in some of these parameters could indicate the activation of TRHergic neurons and their possible involvement in some memory related process. Male Wistar rats were immersed (10 times) for 1, 3 or 5 days in a Morris water-maze containing, or not (yoked control) a platform and sacrificed 5, 30 and 60 min after last trial. TRH content and TSH serum levels were determined by radioimmunoassay; mRNA levels of pro-TRH, TRH-R1, TRH-R2, and PPII, by RT-PCR. Exclusive changes due to spatial training were observed in posterior hippocampus of rats trained for 5 days sacrificed after 60min: decreased TRH content and increased mRNA levels of pro-TRH and TRH-R1, particularly in CA3 region (measured by in situ hybridization). The hypothalamus-pituitary axis responded in both yoked and trained animals (increasing serum TSH levels and pro-TRH expression, due to swim-stress); in the amygdala of both groups, pro-TRH expression increased while diminished that of both receptors and PPII. Differential expression of these parameters suggests involvement of TRH hippocampal neurons in memory formation processes while changes in amygdala could relate to TRH anxiolytic role. The differential modulation in anterior and posterior portions of the hippocampus is discussed.

Ontogeny of prolyl endopeptidase and pyroglutamyl peptidase I in rat tissues.

Prolyl endopeptidase and pyroglutamyl peptidase I are enzymes which participate in the degradation of thyrotropin-releasing hormone (TRH), a hormone which is thought to play an important role in the development of organs and tissues. Here, we have characterized the ontogeny of TRH degrading enzyme activity in the brain cortex, lung, heart, kidney and liver. Overall, prolyl endopeptidase activity was found to be 2 to 5 fold higher in newborn vs. adult rat tissues, with the exception of the soluble form in the liver and the particulate form in the lung. In contrast, the developmental profile of pyroglutamyl peptidase I activity was found to be more variable and tissue dependent. These results corroborate the idea that both enzymes play important, tissue-specific roles during the development and maturation of rat organs.

Cloning and heterologous expression of bovine pyroglutamyl peptidase type-1 in Escherichia coli: purification, biochemical and kinetic characterisation.

We describe the cloning, expression and purification of the bovine XM866409 form of pyroglutamyl peptidase type-1 (PAP1). The cloned nucleotide sequence has an ORF coding for a primary sequence of 209 amino acid residues, which displays 98% identity with the human AJ278828 form of the enzyme. Three amino acid residues at positions 81, 205 and 208 were found to vary between the two sequences. The recombinant bovine PAP1 with a C-terminal His(6) tag (rBtaPAP1(6H)) was expressed in Escherichia coli XL10-Gold cells and purified by immobilised nickel ion affinity chromatography resulting in a yield of 2.6 mg of PAP1 per litre of culture. Purified rBtaPAP1(6H) had a specific activity of 3633 units mg(-1). SDS-PAGE revealed a band for bovine PAP1 with a molecular weight of approximately 24 kDa, which is in good agreement with previously reported data on PAP1. The K (m) and k (cat) values obtained for rBtaPAP1(6H) were 59 muM and 3.5 s(-1), respectively. The optimum pH for activity was 9.0-9.5 and the optimum temperature was 37 degrees C. rBtaPAP1(6H) was found to have an absolute requirement for the thiol-reducing agent DTT, consistent with the expected property of a cysteine protease. Kinetic studies using the peptides pGlu-His-Pro-NH(2) (TRH), pGlu-Ala and pGlu-Val revealed K (i) values of 44.1, 141 and 652.17 microM, respectively. The lowest K (i), observed for Thyrotropin-releasing Hormone (TRH), indicates that rBtaPAP1(6H) has a higher affinity for tripeptides over dipeptides.

Applications of microwave-assisted proteomics in biotechnology.

Biotechnology has recently celebrated 30 years both as a science and as a multi-billion dollar industry. One application of biotechnology is to use human genetic information to discover, develop, manufacture, and commercialize biotherapeutics. Recombinant proteins can be produced in large quantities at high purity. High-throughput proteomic analysis is at the heart of the biotechnology research and development process, and the industry is constantly striving to streamline and automate the analytical processes involved. Microwave-assisted proteomics has recently emerged as a tool for increasing the bio-catalysis of several processes including tryptic digestions lipase selectivities, identification of metal-catalyzed oxidation sites on proteins, identification of protein N- and C-termini and enzyme catalyzed N-linked deglycosylation. Here, we explore the above mentioned methods, and describe our experiences evaluating microwave-technology for other common proteomic protocols including: removal of N-terminal pyroglutamyl for antibody characterization, beta elimination and Michael addition for identification of phosphorylation sites on recombinant proteins and enzyme mediated O-linked deglycosylation.

Differentiation of Leishmania major is impaired by over-expression of pyroglutamyl peptidase I.

Pyroglutamyl peptidases I (PPI) are cysteine peptidases of the clan CF, family C15, which hydrolyse N-terminal l-pyroglutamyl residues (l-pGlu). The l-pGlu modification is a post-transcriptional modification that confers relative aminopeptidase resistance and, in some cases, is essential to the modified peptides' biological activity. PPIs have been identified in a variety of organisms, although definitive biological functions have yet to be attributed to them. The L. major PPI was expressed in Escherichia coli as active recombinant enzyme, and shown to have biochemical properties more similar to mammalian than bacterial PPIs. The LmPPI active site catalytic triad of E101, C210, and H234 was confirmed by mutagenesis. PPI activity was detected in L. major promastigotes, and the enzyme localised to the parasite cytosol. No detectable phenotype could be observed for L. major PPI-deficient mutants, which retained infectivity to macrophages in vitro and mice. However, over-expression of the active PPI, but not inactive PPI(C210A), in L. major impaired differentiation from the procyclic promastigote to the infective metacyclic promastigote. Susceptibility to a natural l-pGlu-modified antimicrobial peptide, gomesin, was tested using the different cell lines, which were all equally susceptible. Whilst PPI is widespread through the eukaryotic kingdom, this study now suggests that the enzyme is not essential for normal eukaryotic cell function. However, PPI could be involved in regulating the action of l-pGlu-modified peptides required for differentiation of L. major.

Unfolding of the immunoglobulin light and heavy chains is required for the enzymatic removal of N-terminal pyroglutamyl residues.

To enable Edman sequencing of pyroglutamylated immunoglobulins, enzymatic deblocking by pyroglutamate aminopeptidase is performed, often with variable yield and compromised solubility. Recently, enzymatic deblocking of immunoglobulins without denaturation was described. Although the conditions ensured efficient removal of pyroglutamyl residues, we conclude that deblocking is preceded by denaturation, which results in aggregation of the immunoglobulins. To study the effect of folding status on deblocking we developed a methanol based deblocking solution, which preserved the enzymatic activity of pyroglutamate aminopeptidase, provided conditions compatible with sequencing and enhanced deblocking of electroblotted samples, as well. At 50 degrees C and 35% (v/v) methanol the immunoglobulin chains were completely aggregated, but the degree of deblocking was comparable to that obtained with the previously described method. At 37 degrees C, the immunoglobulins were partly aggregated, but the deblocked chains were completely in the insoluble fractions, whereas the soluble fractions had retained pyroglutamylation in both chains, suggesting that unfolding of the immunoglobulins is required for the excision of the pyroglutamates. Inspection of the structures of pyroglutamylated immunoglobulin and pyroglutamate aminopeptidase P. furiosus indicates that the enzyme requires the substrate in an extended conformation, a criterium, which we conclude not to be fulfilled in the native form of immunoglobulins. Unfolding of the N-terminus would disrupt the immunoglobulin fold by breaking interactions between secondary structure elements and expose surfaces prone to aggregation.

Occurrence of the free and Peptide forms of pyroglutamic acid in plasma from the portal blood of rats that had ingested a wheat gluten hydrolysate containing pyroglutamyl peptides.

In order to determine pyroglutamic acid levels in plasma, we developed a method based on precolumn derivatization of the carboxyl group of pyroglutamic acid with 2-nitrophenylhydrazine. Eight-week-old male SD strain rats were administered 200 mg of an acidic peptide fraction obtained from a commercial wheat gluten hydrolysate containing 0.63 mmol/g pyroglutamyl peptide. After administration, significant amounts of free pyroglutamic acid were observed in the ethanol-soluble fraction of the plasma from the portal vein. In addition, pyroglutamate aminopeptidase digestion of the ethanol-soluble fraction liberated significant amounts of pyroglutamic acid, which indicated the presence of the pyroglutamyl peptide. The presence of the pyroglutamyl peptide in the plasma was further confirmed by size exclusion chromatography. The levels of free and peptide forms of pyroglutamic acid increased significantly and reached a maximum (approximately 40 nmol/mL) at 15 and 30 min after administration, respectively.